The HIV and SIV envelope glycoprotein complex (gp120/gp41) promotes viral entry by mediating the fusion of viral and cellular membranes. The native, trimeric gp120/gp412 complex on the viral surface is a vulnerable target for neutralizing antibodies. A major hurdle in the development of envelope protein immunogens for vaccines is the lack of effective approaches to the production of a stably folded envelope glycoprotein complex due to the labile association between gp120 and gp41 ectodomain. Interestingly, an in-vitro derived variant of SIVmac251, termed CPmac, exhibits an unusually stable gp120-gp41 association, and thus unique phenotype is due to mutations in the gp41 ectodomain. The long-term objective of this research plan is to contribute to the development of an understanding of the physical basis for the gp120-gp41 interaction. The focus of this project is on defining the structural determinants of the stable gp120-gp41 association in CPMAC and translating these conformational elements to the HIV envelope glycoprotein. Specific aims of the proposed research are: (1) To determine the high-resolution crystal structure of the CPMAC gp41 ectodomain core, identify the mutations in this core that are responsible for destabilizing the six-helix bundle structure of fusion- active gp41, and characterize the structural and physicochemical properties of mutant gp41 ore models, using techniques including X-ray crystallography, NMR spectroscopy, and thermodynamic analysis. (2) To characterize the role of CPMAC-like mutations in delineating the folding, thermodynamics, and conformation of gp41 core models from primary HIV isolates, in order to understand structural determinants for strengthening the gp12-[gp41 interaction with the ultimate aim of developing antigenic mimics of the virion-associated envelope glycoprotein complex for functional antibody induction. (3) To identify stable, folded subfragments of the CPMAC envelope glycoprotein by protein dissection in an effort to determine the structure of the gp120/gp41 complex by X-ray crystallographic methods. The proposed research will increase our knowledge about structure/function relationships of the HIV-1 envelope glycoproteins. These studies will also be crucial for guiding our efforts to target the HIV-1 entry process for vaccine and anti-viral drug development.